COMPOSITIONS AND METHODS EMPLOYING UNIVERSAL-BINDING NUCLEOTIDES FOR TARGETING MULTIPLE GENE VARIANTS WITH A SINGLE siRNA DUPLEX

Abstract

Provided are siRNA molecules of between about 15 base-pairs and about 40 base-pairs comprising one or more universal-binding nucleotide such as inosine, 1-β-D-ribofuranosyl-5-nitroindole, and 1-β-D-ribofuranosyl-3-nitropyrrole, compositions comprising one or more universal-binding nucleotide comprising siRNA, and methods for making and for using such universal-binding nucleotide comprising siRNA molecules to increase the specific binding of the modified siRNA molecule to variants of a target sequence such as, for example, when in contact with a biological sample and to reduce off-target effects of the siRNA molecule.

Description

[0001]This patent application claims priority under 35 U.S. § 119(e) of U.S. Provisional Application No. 60 / 796,274 filed Apr. 27, 2006, the contents of which are incorporated herein by reference.BACKGROUND OF THE DISCLOSURE[0002]1. Technical Field of the Invention[0003]The present invention relates to the treatment of disorders by means of RNA interference (RNAi). More specifically, the present disclosure relates to the targeted delivery of small inhibitory nucleic acid molecules (siRNA) that are capable of mediating RNAi against genes, and variants thereof, wherein the siRNA comprise one or more universal-binding nucleotide such as, for example, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and 1-β-D-ribofuranosyl-3-nitropyrrole.[0004]2. Description of the Related Art[0005]RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small inhibitory nucleic acid molecules (siRNAs) a double-stranded RNA (dsRNA) that is homolog...

AI Technical Summary

Benefits of technology

[0016]The present disclosure fulfills these and other related needs by providing compositions and methods for increasing the number of target RNAs, such as variants of viral RNAs or endogenous genes, that are susceptible to degradation facilitated by one or more small inhibitory nucleic acid(s) (siRNA(s)). Compositions described herein incorporate one or more universal-binding nucleotide(s) in a first, second, and / or third position in an anti-codon of an anti-sense strand of an siRNA duplex thereby increasing the number of RNA to which the siRNA anti-sense strand specifically binds.

[0021]Within certain embodiments, the isoleucine anti-codon UAU, for which AUA is the cognate codon, may be modified such that the third-position uracil (U) nucleotide is substituted with the universal-binding nucleotide inosine (I) to create the anti-codon IAU. Inosine is an exemplary universal-binding nucleotide that can nucleotide-pair with an adenine (A), uracil (U), and cytosine (C) nucleotide, but not with a guanine (G). This modified anti-codon IAU increases the specific-binding capacity of the siRNA molecule and thus permits the siRNA to pair with mRNAs having any one of AUA, UUA, and CUA in the corresponding position of the coding strand thereby expanding the number of available RNA degradation targets to which the siRNA may specifically bind.

[0031]It will be understood that, regardless of the position at which the one or more universal-binding nucleotide is substituted, the siRNA molecule is capable of binding to a target gene and one or more variant(s) thereof thereby facilitating the degradation of the target gene and / or variant thereof via a RISC complex. Thus, the siRNA of the present disclosure are suitable for introduction into cells to mediate targeted post-transcriptional gene silencing of a target gene and / or variants thereof.

[0032]Within still further aspects of the present disclosure are provided methods for selecting modified siRNA molecules that are capable of specifically binding to a wide range of desired gene target variants while being incapable of specifically binding to non-desired gene target variants. The selection process disclosed herein is useful, for example, in eliminating modified siRNAs that are capable of exerting a cytotoxic effect resulting from non-specific binding to, and subsequent degradation of, one or more non-target genes.

Problems solved by technology

This approach, however, employs a single RNA target methodology and ignores the possibility that variants of the target RNA (gene variants) may be present or later developed within the cell.

Thus, the introduction of an siRNA with a specific nucleotide sequence may target a particular mRNA for destruction, yet remain ineffective in destroying variants of that RNA.

In this context, a particular siRNA that targets a specific viral RNA may initially function as a therapeutic agent, but due to the rapid mutation rate of the viral gene, lose the ability to target the viral RNA for degradation.

Thus, there remains a long-standing unmet need in the art for compositions and methods that improve the effectiveness of siRNA-mediated gene silencing.

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